Chronic Exposure to Biomass Ambient Particulate Matter Triggers Ams Polarization and Activation in Rat Lung

Background: Chronic Obstructive Pulmonary Disease (COPD) is a chronic inammatory disease in which a variety of immune cells are involved; among them, the role of alveolar macrophages (AMs) remains unknown in the pathogenesis and development of COPD. We aimed to study the function of AMs from different stages of chronic biomass fuel (BMF) exposed rats, and investigate the signal pathways which regulate AMs polarization. Methods: 180 male Sprague-Dawley rats were divided into BMF group and clean air control (CON) group. After BMF exposure for 4 days, 1 month, and 6 months, the cytokine secretion and function of AMs were determined by ow cytometry, and further conrmed in mRNA level by qPCR, in protein level by WB and immunouorescent assay. Bone marrow derived macrophages(BMDMs) were cultured, and PPARγ agonist, PPARγ KO lentivirus, and TGFβ1were used as the intervention in vitro. Results: We found that pro-inammatory factors increased, while CD206 in AMs decreased remarkably at 4 days. Interestingly, pro-inammatory macrophages shared a feature of anti-inammatory macrophages. Consistent with IL4 upregulated in BALF, p-Stat6 and PPARγ in AMs elevated at 4 days. After BMF exposure for 6 months, CD206, TGFβ1, and p-Smad3 were signicantly higher than the control groups. PPARγ reversed the M1 phenotype induced by PM, and drove the macrophages into the M2 phenotype in vitro. Conclusion: We demonstrated that the dynamic phenotype and functional change of AMs during BMF exposure, and both PPARγ and TGFβ1 were important molecules regulated AMs’ function. polarization and participating in lung tissue remodeling in BMF models. Our study provided the dynamic phenotype and functional change of AMs during BMF exposure, suggesting AMs play a pro-inammatory role in the early stage and an anti-inammatory role associated with promoting tissue remodeling in the latter stage of COPD. Further identication of a few signal pathway molecules involved in AMs polarization under BMF exposure, may provide potential targets for the COPD treatment.


Background
Chronic Obstructive Pulmonary Disease (COPD) seriously endangers human health, with a high disability rate and fatality rate, causing a huge economic and social burden. According to an epidemiological study in China, the prevalence of COPD among people over 40 years old has increased to 13.7% 1 . Factors that in uence COPD development and progression are extremely complex, and smoking is not the only factor.
Almost 3 billion people worldwide use biomass wood as the main source of energy for cooking, heating, and other household needs. Indoor biomass smoke exposure increases the risk of COPD. Moreover, a high level of biomass smoke exposure accounts for 50% of deaths in patients with COPD in developing countries 2 , and is associated with the increase of COPD hospitalization [3][4][5][6][7] . Improvements in cooking fuels and kitchen ventilation had e cient effects on the decline in FEV 1.0 8 . Although the pathologic features of COPD patients induced by biomass ambient particulate matter are less severe than those of smoking COPD patients 9 , its danger should not be ignored. However, the molecular pathway of pathological damage caused by biomass ambient particulate matter in vivo and vitro remains less understood.

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COPD is characteristic of a mixture of small airway disease and parenchymal destruction. Most research focused on airway in ammation. Recently studies have linked changes to host defense and lung immunity. Alveolar macrophages form the rst line of immune defense in lung tissue and play an important role in maintaining lung local immune homeostasis. They contact outside air constantly, and perform their pattern recognition receptors to recognize invading pathogens and initiate the in ammatory response. When the dangerous substances are cleared, macrophages can secrete anti-in ammatory mediators and growth factors to promote the elimination of in ammation and tissue repair. Immune phenotype and function of AMs are greatly affected by the local microenvironment of the alveolar lumen.
Macrophages can be classi ed into classical activated M1 macrophage and alternative activated M2 macrophage 10 . When stimulated by Th1 cytokines such as interferon -γ (IFN-γ) and toll-like receptor signaling, M1 activates 11 . M1 expresses CD86, secretes IL1 and TNFα, and has a strong ability to kill pathogenic microorganisms 12 . M1 secretes IL6, IL12, and IL23 to promote the differentiation of Th1 and Th17 cells and promote the in ammatory response 13 . The signal pathway molecules include Stat1, NFκB, and mitogen-activated protein kinases(MAPKs). The antigen presentation capacity of AMs is very weak, which helps the macrophages not respond to harmless pathogens and reduce the release of in ammatory cytokines 14,15 . When stimulated by Th2 cytokines such as IL4 and IL13, M2 activates and expresses CD206, CD163, TGFβ, tyrosine-protein kinase, arginase 1, Stat6, and Stat3, which are involved in tissue repair and anti-in ammatory effects 13,16,17 . M2 also secretes EGF and VEGF growth factors to promote tissue repair 17  AMs increased in COPD patients, and released a number of in ammatory mediators to contribute to pathology 21,22 . In contrast, it's reported that AMs in BALF of COPD patients were the high expression of M2 markers 23 . But Bazzan E and co-workers have shown that both iNOS and CD206 were expressed by macrophages in the lungs of non-COPD smokers and COPD patients, indicated that macrophages in the lungs were polarized bidirectionally 24 . The function of AMs in COPD is controversial. Diversity and plasticity are characters of macrophages in vivo 25 . In order to maintain the homeostasis of the body, immunity is always in a dynamic change during the occurrence and development of the disease, and so do AMs. In the lung of animal models exposed to nitrogen mustard, M1 macrophages were predominant at 1-3 days, while M2 macrophages appeared at 28 days 26 . Another study also showed M1 and M2 macrophages were activated sequentially in the lung after exposure of carbon nanotubes, which caused pulmonary injury progressing to brosis 27 . But there is no research about the dynamic change of AMs' immune function in pathogenesis and development of COPD.
In this study, by establishing the biomass ambient particulate matter exposed rat model, we showed phenotypic change and immune function of AMs in vivo. To gain a better understanding how alveolar macrophage switched in COPD progress, we also investigated the dynamic change and function of some transcriptional factors for AMs activation.

Methods
Animals. 180 male Sprague-Dawley rats (170-200g, 6-8weeks old) were housed in the laboratory animal center of Guangzhou Medical University in barrier condition. The rats were randomly divided into biomass fuel (BMF) group and clean air control (CON) group. The experimental protocol and animal care was in compliance with the guiding principles for the care and use of laboratory animals recommended by the Chinese Association for Laboratory Animal Science Policy. Guangzhou Medical University Animal Research Ethics Board approved all experiments. The animal facility maintained a 12 hours light/dark cycle, and room temperature was xed at 20 ± 2°C with 45-65% relative humidity.
BMF Exposure System. Rats were exposed to smoke produced by smoldering China r saw-dust (2g/per heating panel/per time) for two 2-hours periods, 5 days per week, for 4 days, 1 month and 6 months. The BMF exposure system primarily consisted of a wood-burning unit and a whole body exposure unit. The size of the animal exposure chamber was 265×205×140mm(L×W×H). The BMF was generated by eight heating panels (500w), which were connected in series in wood-burning chamber. Each heating panel had worked for 20 minutes, then the next heating panel started. Biomass fuel smoke was set into the animal exposure chamber. Besides, there were two sampling ports to monitor various characteristics of exposure PM and gas in exposure chamber.
PM collection and extraction. PM was collected from the burning of China r during high-temperature combustion with moderate air supply(April 23-May 6, 2015) in accordance with the procedures described previously 28 . High volume sampler (TE-6070, Tisch, USA) equipped with a PM2.5 selective-inlet head(1.13m 3 /min) was used to collected particle. PM was collected on the ber membrane lters with 1.6µm pore size for up to 2 hours. Exposed lters were soaked in water for 10 min and then in dichloromethane for 4 hours. The extracted solution was lyophilized and mixed. The weight of PM was de ned as increase amount for each lter. The PM sample was dissolved in DMSO to 100mg/ml, and then diluted with culture medium to provide a concentration of less than 0.01% DMSO.
Sample preparation and isolation of AMs. Rats were sacri ced after 4 days, 1 month, and 6 months exposure period. Bronchoalveolar lavage uid (BALF) was conducted by instilling the lungs sequentially with 8ml ice cold PBS for 4 times. BALF was centrifuged to obtain the cells and supernatants. The cells were suspended with 1ml PBS and counted with a cell counter (Millipore Scepter2.0, USA).
Bone marrow-derived macrophages culture and stimulation. Bone marrow cells were obtained from the femur and tibia bones, and were incubated in RPMI-1640 medium supplemented with 10% heatedinactivated fetal bovine serum and recombinant rat GM-CSF (10ng/ml, Peprotech) at 10ng/ml for 7days as described 29 . PM was used at a concentration ranging from 0 to 45µg/ml. Rosiglitazone (1µM, Sigma), IL4(50ng/ml, Peprotech) and TGFβ1(10 ng/ml, Peprotech) were added 1 hour before PM treatment. WB. Lung tissue was lysed by using RIPA buffer (Thermo Scienti c). Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane(BIO-RAD). The membrane was blocked with 5% BSA ( Histological staining. The lavaged lung (left lung) was then in ated with 4% formaldehyde, and maintained at a pressure of 25cmH 2 O to keep for histological assessment. Sections (5µm) were measured with hematoxylin and eosin (H E) staining to investigate lung morphometric change.
Statistical analysis. Statistical analyses were performed by IBM SPSS 22.0, and data were expressed as mean ± SD. Two group comparisons were accomplished by an unpaired t-test. More than two groups comparisons were analysed using one-way ANOVA test. Mann-Whitney U-tests were used to compare relative mRNA expression and CD206 MFI between experimental groups. p 0.05 was considered signi cant.

Results
Determination of particle size distributions and gas concentrations in the exposure chamber. To measure the particle size distributions in suspension and gas concentrations, we used Dust Trak aerosol detector (TSI, Shoreview, USA) and smoke Test340 portable gas analyzer (Testo, Lenzkrch, Germany) to evaluate the quality control parameters of the exposure system. The value of PM 1 , PM 2.5 , and PM 10 were 27.77 ± 8.66 mg/m 3 , 28.07 ± 8.84 mg/m 3 , and 28.23 ± 8.86mg/m 3 in the BMF exposure room, respectively (Supplementary Table 2). The CO concentration was maintained at a low level of 55.16 ± 13.77 ppm, and NO and SO 2 weren't detected.
BMF induced Lung morphological changes and AMs in ltration. In order to investigate whether air pollution matter exposure causes emphysema in our exposed rat model, we did hematoxylin and eosin (H E) staining to examine lung morphometric character. Alveolar enlargement was calculated as the mean linear intercept(MLI), and the bronchial wall thickness was quanti ed by wall thickness = (total bronchial area-lumen area)/total bronchial area. Our data showed that BMF exposure induced emphysematous changes and airway remodeling ( Fig. 1a-d). Long-term BMF exposure damaged the lung parenchyma and airway wall, which led to alveolar enlargement and distal airway remodeling. Histological analysis demonstrated that the airway wall thickness increased (p 0.01), and the mean linear intercept decreased dramatically (p 0.01) at 6 months, whereas, there was no change at 1 month compared to controls (p = 0.366 and 0.557). Total BALF cells in BMF exposure groups were increased compared with the control groups after 4 days, 1 month, and 6 months BMF exposure (Fig. 1e, p = 0.013, 0.001, and 0.003, respectively). AMs were labeled with pan macrophage surface marker CD68, and de ned as CD68 + subpopulation with the purity displayed as a percentage of parent population gated on FSC-A/SSC-A. The numbers of in ltrated AMs were more than CON groups after 4 days, 1 month, and 6 months BMF exposure (Fig. 1f, p = 0.01, 0.04, and 0.003, respectively), and reached a peak at 6 months of BMF exposure.
Phenotypic characterization of AMs polarization induced by BMF exposure. In order to investigate gene expression of AMs when exposed to BMF, we also used quantitative PCR to determine the mRNA expression for a few key genes (Fig. 3). The result showed that iNOS and IL1β signi cantly ascended at 4 days of BMF exposure (Fig. 3a, p = 0.005 and 0.001), and descended to near normal levels during the subsequent time. TNFα moderately elevated in 1 month BMF exposure (Fig. 3b, p = 0.028), and declined in 6 months BMF exposure (Fig. 3c, p = 0.374), consistent with BALF cytokines expression. Whereas, TLR2 and TLR4 had no change during the whole exposure course, consistent with previous studies 30,31 . The level of EGF mRNA was upregulated in AMs of rats exposed to 4 days BMF (Fig. 3a, p 0.01), which was consistent with the level of EGF protein expression in BALF.
To further investigate the effect of BMF exposure on the dynamic phenotype change of AMs in rats, in addition to the gene expression, we assessed the CD206 (M2 marker) and CD86 expression (M1 marker) in AMs (Fig. 3). The result showed that CD206 MFI decreased at 4 days of BMF exposure (Fig. 3d,e, p 0.01), and increased to near controls following 1 month exposure(p = 0.207), and was signi cantly higher than the control group following 6 months exposure(p = 0.035). Conversely, CD86 MFI had no change in AMs during the whole exposure of biomass fuel smoke (Fig. 3f,g, p = 0.730, 0.831, and 0.995, respectively). The result indicated that BMF exposure reduced the anti-in ammatory marker expression in AMs at the beginning, and the anti-in ammatory marker expression was increasing with the accumulation of exposure time.
BMF exposure triggered signaling pathways of macrophage polarization and activation. To study which signaling pathways involved in AMs polarization and activation under the BMF exposure, especially those involved in M2 polarization to attenuate the in ammatory response and promote tissue remodeling 13,32 , such as Stat6, Stat3, PPARγ, and TGFβ1. We used quantitative PCR, western blot, and immuno uorescence to determine the mRNA and protein level of Stat6, Stat3, PPARγ, and TGFβ1 in BMF exposed rats. It showed that Stat6 mRNA expression in AMs increased signi cantly after 4 days of BMF exposure (Fig.S1a, p < 0.01), and descended to near controls during the subsequent time (Fig.S1b,c, p = 0.149 and 0.661). The level of p-Stat6 increased after 4 days of BMF exposure (Fig. 4a,b, p < 0.01). Stat3 mRNA expression in AMs had no change compared to control groups after 4 days, 1 month, and 6 months of BMF exposure (Fig.S1a,b,c, p = 0.112, 0.209 and 0.832). In contrast, the level of p-Stat3 level elevated after 4 days of BMF exposure (Fig. 4a,b, p = 0.003), and then declined to near control group after 1 month and 6 months exposure (Fig. 4a,b, p = 0.898 and 0.484). PPARγ mRNA expression in AMs elevated at 4 days of BMF exposure (Fig.S1a, p < 0.01), and declined to a normal level during the subsequent course (Fig.S1b,c, p = 0.66 and 0.543). There was no change of PPARγ protein in lung tissue between controls and exposure groups after 1 month and 6 months of BMF exposure (Fig. 4a,b, p = 0.934 and 0.572). But, PPARγ protein signi cantly increased at 4 days of BMF exposure (Fig. 4a,b, p = 0.005), consistent with PPARγ mRNA expression in AMs.
PPARγ primed BMDMs exposed to PM into alternative macrophages. To study whether PPARγ reversed M1 phenotype induced by biomass ambient particulate matter, and drove the macrophages into M2 phenotype, we cultured bone marrow derived macrophages(BMDMs), and stimulated them with PM extracted in our lab, and used PPARγ agonist and PPARγ KO lentivirus as the intervention in vitro. 30µg/ml PM was selected as an intervention concentration (Fig.S2a-c). The effect of PPARγ on in ammatory factors was determined via quantitative PCR, western blot, and immuno uorescence. 30µg/ml PM induced BMDMs to secrete iNOS, IL1β, TNFα, and TLR2 (Fig. 6a, p < 0.01 for all genes), and BMDMs showed a pro-in ammatory phenotype. Transcriptional factor PPARγ inhibited pro-in ammatory genes (Fig. 6a, p < 0.01 for all genes). 30µg/ml PM triggered phosphorylation of IKBα from 6 hours to 12 hours, and triggered p-P65 began to rise after 6 hours (Fig.S3a,b). PPARγ overexpression signi cantly inhibited phosphorylation of P65 and IKBα (Fig. 6b, p < 0.01 for all comparisons). PPARγ overexpression also repressed upregulation of IL1β protein level induced by PM (Fig. 6b). PPARγ KO lentivirus increased the level of p-P65 and p-IKBα (Fig. 6c, p < 0.01 for all comparisons). Immuno uorescence staining showed that PPARγ KO lentivirus promoted p-P65 nucleus translocation, while PPARγ overexpression inhibited p-P65 nucleus translocation induced by PM (Fig. 6d).

Discussion
Indoor air pollution induced by biomass ambient particulate matter is strongly link to incidence and hospitalization rates of COPD. Our previous study showed that biomass ambient particulate matter retention in lung tissue induced pulmonary in ammation, airway remodeling, and alveolar cavity enlargement. The chronic BMF exposure model serves as a useful model to analyze how indoor air pollution promotes the progress of emphysema in lung. In this work, we demonstrated that the emergence of pro-in ammatory macrophage eventually conversed into ant-in ammatory macrophage associated with BMF induced in ammation, which revealed that biomass ambient particulate matter initiated this plasticity in AMs. In addition, functional conversion of AMs was regulated by signal pathways. We also found the dynamic change of some regulated molecules, which drove AMs into an anti-in ammatory phenotype.
Airway in ammation, airway remodeling, and alveolar cavity enlargement were observed in our BMF exposure model. Our data showed that the early stage of BMF exposure models was characterized as airway in ammation, and the later stage was characterized by airway remodeling and alveolar cavity enlargement, which was in agreement with previous observation in COPD models 33 .Our previous study also showed that PEF and FEV(20)/FVC in 7 months exposure group were signi cantly lower than the control group, indicating that BMF induced dysfunction of lung function 33 . CD68 is a speci c macrophage marker in rats, mice, and humans, and F4/80 is expressed in mature macrophages in mice.
CD11b is expressed in both rats and mice, but is low expressed in AMs 34 . In this study, we used CD68 as a marker of AMs. Our data showed that BMF exposure induced macrophages in ltration in BALF, and the number of AMs increased with the accumulation of exposure time.
Surprisingly, molecular analyses revealed that the emerging pro-in ammatory macrophages shared a feature of anti-in ammatory phenotype, which was partially overlapping but also distinct, including the co-expression of mRNA encoding of IL1β, iNOS, and PPARγ. The result also indicated that AMs produced pro-in ammatory factors to damage lung tissue and subsequently skewed towards anti-in ammatory phenotype after short-term exposure. After 6 months of BMF exposure, the protein level of TGFβ1 increased, and airway and lung tissue were remodeled, which resulted in the COPD pathology. The whole course from pro-in ammatory phenotype to anti-in ammatory phenotype, and then to the chronic pathology, indicated an interaction between BMF exposure and the pulmonary immune system.
In ammation happened in the early stage of BMF exposure. Camila Oliveira da Silva and co-workers observed a dynamic change of cytokine production in CS exposure mice. They found that TNFα and NO increased in ve days CS exposure mice, and then greatly decreased in 14 days of CS exposure.
Afterward, 30 days of CS exposure increased TGFβ1 production in the lung 35 . We also found IL1α, IL1β, IL12p70, LIX as well as IL4 increased remarkably in BALF exposed to short-term biomass fuel smoke.
IL1α/β, which are mainly produced by activated monocytes and macrophages, enhance B cell proliferation and maturation, NK cytotoxicity, pro-in ammatory chemokine expression, as well as acute phase protein expression. IL12p70, produced by monocytes and macrophages, can further act on lymphocytes, and effectively promote Th1 response in COPD. LIX, which is a small cytokine of the CXC chemokine family, is produced by epithelial cells following stimulation with IL1 or TNFα, and promotes the chemotaxis of neutrophils. Pro-in ammatory cytokine dynamic change revealed that the most severe in ammatory injury is in the early stage of exposure models. Interestingly, Th2 cytokine IL4 also increased simultaneously, which was able to promote AMs towards anti-in ammatory phenotype. Then the pro-in ammatory cytokines gradually decreased during the subsequent exposure course. The increase of IL4 and the recovery of in ammation didn't match in time in COPD models, which was in conformity with the previous study in acute exacerbation of chronic obstructive pulmonary disease 36 .
Previous studies have reported that M2 secreted EGF and VEGF to promote tissue repair 17,37 . The data suggested that EGF and VEGF upregulated under the action of Th2 cytokine in the early stage of BMF exposure. Renat Shaykhiev and co-workers provided transcriptome-base evidence that smoking induced reprogramming towards M2 polarized macrophage in COPD patients for the rst time, suggesting that AMs were likely involved in COPD pathogenesis in a non-in ammatory manner 38 . However, the clinical study could not track the change of immune cells on the time axis. Our study provided the dynamic phenotype and functional changes of AMs during BMF exposure.
Macrophage polarization is controlled by signal pathway molecules. Which signal pathways regulate macrophages have yet to be identi ed in the COPD model. In the present study, anti-in ammatory phenotype took the most time of BMF exposure period, and overlapped with pro-in ammatory phenotype at 4 days of BMF exposure. We focused on signal pathways of anti-in ammatory phenotype in the BMF exposure model more. IL4 skews macrophages toward the M2 phenotype, and activates Stat6 and Stat3. Stat6 modulates many genes associated with the M2 phenotype, including CD206, arginase 1, and resistin likeα 39 . The present study found phosphorylation of Stat6 and Stat3 increased after 4 days of BMF exposure compared to controls, consistent with upregulation of IL4. It supported that Stat6 and Stat3 signals participated in activation of M2 in the early stage.
PPARγ is one of the important transcription factors of M2, and primes monocytes into alternative activated macrophages 20,40 . The dynamic change of PPARγ has not been reported in previous studies. Our data showed that PPARγ elevated during the most severe in ammation periods, and then returned to the normal level as in ammation disappeared. We further demonstrated that PPARγ was upregulated in BMDMs by IL4 in vitro. The Th2 cytokine IL4 was required for the development of M2, and IL4 mediated signals stimulated PPARγ expression 20,41,42 . In addition, our study showed that PPARγ signi cantly inhibited activation and nucleus translocation of NFκB p65 via repression of IKBα phosphorylation, and upregulated M2 markers CD206 and p-STAT6 expression, suggesting PPARγ reversed M1 phenotype induced by PM, and drove the macrophages into M2 phenotype. PPARγ rapidly drove the transformation of AMs into anti-in ammatory phenotypes, and played a protective role in the short-term of BMF exposure in the study. There are some drugs in the PPARγ pathway, which provide a potential target for intervention in COPD. Simon Lea and co-workers found that rosiglitazone inhibited cigarette smoke-induced pulmonary in ammation 43 . Rosiglitazone was able to reduce exacerbations through attenuating pulmonary in ammation and decreasing bacterial burden 44 . As a result, PPARγ may be an effective approach for the treatment of COPD.
TGFβ1 is another important regulatory molecule of M2. TGFβ1 is a critical cytokine for the development and maturation of AMs. TGFβ1 also repressed macrophage-derived in ammatory gene 45,46 . M2 participates in the development of brosis and contributes to disease pathogenesis. M2-derived TGFβ1 promoted tissue remodeling and wound repair by blocking the extracellular matrix's degradation and eliciting synthesis of interstitial brillar collagens 13 . Besides, it's con rmed that airway remodeling via the TGFβ1 pathway leads to the thickening of the small airway wall. TGFβ1 in AMs may be involved in the mechanism of airway remodeling. Our study showed that AMs maintained an anti-in ammatory phenotype characterized by elevated CD206 and TGFβ1 markers without stimulation of Th2 cytokines at 6 months of BMF exposure. We also found that TGFβ1 promoted CD206 expression in BMDMs in vitro.
Chronic BMF exposure induced TGFβ1 production, and activated the downstream signal Smad3 involved in tissue remodeling. Activation of TGFβ1 in the late stage of BMF exposure, indicated the role of TGFβ1 in regulating M2 polarization and participating in lung tissue remodeling in BMF models.

Conclusion
Our study provided the dynamic phenotype and functional change of AMs during BMF exposure, suggesting AMs play a pro-in ammatory role in the early stage and an anti-in ammatory role associated with promoting tissue remodeling in the latter stage of COPD. Further identi cation of a few signal pathway molecules involved in AMs polarization under BMF exposure, may provide potential targets for the COPD treatment.   BMF altered the expression of genes and surface markers in alveolar macrophages. a iNOS, IL1β, and EGF mRNA expression upregulated in AMs exposed to BMF 4 days. b TNFα mRNA expression in AMs had changed after 1 month of BMF exposure. c iNOS, IL1β, TNFα, TLR2, TLR4, and EGF mRNA expression had no changed after 6 months of exposure. d,e Comparison of CD206 expression in AMs between BMF and CON groups. f,g Comparison of CD86 expression in AMs between BMF and CON groups. Data represent the mean ±SD of a minimum number of 6 rats per group. *p<0.05, ** p<0.01, signi cantly different from clean air groups. PPARγ protein expression in lung tissue between groups. c Time-dependent activation of p-Stat6 was examined by double Immuno uorescence staining of p-Stat6 (green) and CD68 (red). d Time-dependent activation of PPARγ was examined by double Immuno uorescence staining of PPARγ (green) and CD68 (red). Bars are equal to 20μm. The value in a and b represent the mean ±SD of a minimum number of 6 rats per group. *p<0.05, ** p<0.01, signi cantly different from CON groups. and Smad3 in lung tissue. d TGFβ1 in AMs was examined by double Immuno uorescence staining of TGFβ1 (green) and CD68 (red). e p-Smad3 in AMs was also examined by double Immuno uorescence staining of p-Smad3 (green) and CD68 (red). Bars are equal to 20μm. The value in b and c represent the mean ±SD of a minimum number of 6 rats per group. *p<0.05, ** p<0.01, signi cantly different from CON groups.  PPARγ primed BMDMs into M2 phenotype. a,b Western blotting showed comparison of PPARγ and p-STAT6 expression in BMDMs between groups. c Comparison of CD206 MFI in BMDMs between groups.
The value in b and c represent the mean ±SD of 6 independent experiments. *p<0.05, ** p<0.01, signi cantly different from control groups.  Schematic representation of AMs activation and polarization triggered by BMF exposure. Following 4 days of BMF exposure, pro-in ammatory factors increased, such as iNOS, IL1α, IL1β, and IL12p70, while